Amphetamines (AMPHs) are potent psychostimulants that are widely used and abused, with profound medical and societal impact. They are known to cause mobilization of cytoplasmic dopamine (DA) to the cell exterior via DA transporter (DAT)-mediated efflux, yet the mechanisms that mediate these actions remain poorly defined and are a focus of this proposal. Using heterologous expression systems and a Drosophila behavioral model, we have shown that AMPH-induced DA efflux and consequent behaviors, but not DA uptake, are dependent on N-terminal phosphorylation of DAT. Our team has also made critical advances in understanding the molecular mechanisms of substrate uptake by studying the bacterial transporter LeuT as a prototype, using state-of-the-art single-molecule approaches and computational analyses. Although the N-terminal region is essentially absent in LeuT and was truncated in the Drosophila DAT (dDAT) structures, our team has reported a computational model of the N terminus of the human DAT (hDAT) from ab initio structure prediction in combination with extensive atomistic molecular dynamics simulations. The analysis shows the N terminus to be highly dynamic, to contain secondary structure elements, and to interact with lipid membranes through electrostatic interactions. Here we aim to probe these structural elements to gain insight into the physiology of DAT and its regulation by AMPHs, using our team's synergistic behavioral, biochemical, biophysical, and computational tools. In parallel studies we aim to explore the mechanisms that regulate AMPH-induced release of DA from synaptic vesicles into the cytoplasm. Using multiphoton imaging of living Drosophila brain we have shown that at pharmacologically relevant concentrations, AMPHs must be actively transported both by DAT and by the vesicular monoamine transporter VMAT in order to diminish the vesicular pH gradient and redistribute vesicular contents. Still, how these events lead to redistribution of DA to the cytoplasm remains unknown. Recent data suggest that VMAT N-terminal phosphorylation is essential for AMPH-induced DA efflux from vesicles, and we propose to explore this hypothesis mechanistically and test it in vivo. Our established multi-scale approach integrates biochemistry and biophysics of purified proteins, single-molecule FRET and computational analysis, with cell-based assays, Drosophila brain imaging, analysis of in vivo phosphorylation, and behavioral studies in living flies to probe the role of DAT and VMAT in the actions of AMPHs in the appropriate physiological and structural contexts, in the following SPECIFIC AIMs: AIM 1. To elucidate the role of membrane interactions in modulating phosphorylation of the N terminus of DAT and its ability to mediate AMPH-induced DA efflux and behaviors. AIM 2. To determine how N-terminal phosphorylation alters DAT function and dynamics. AIM 3. To determine the role of VMAT and its putative N-terminal phosphorylation in AMPH-induced DA efflux from synaptic vesicles in vivo and in vitro. This work will provide a clear validation of novel targets for medications that block AMPH action through mechanisms that do not alter DA uptake.